Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other.
Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other.
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DOI：
10.7554/elife.06562
复制
发表时间：
2015-05-13
期刊：
影响因子：
7.7
通讯作者：
Patel SS
中科院分区：
文献类型：
--
作者：
Nandakumar D;Pandey M;Patel SS
前导链DNA合成需要复制解旋酶和DNA聚合酶（DNAP）之间的功能偶联，但对偶联的结构和机理基础知之甚少。本研究以单碱基分辨率定义了T7解旋酶和T7 DNAP在复制叉连接处的精确位置，以创建一个结构模型来解释活动的相互刺激。我们的2-氨基嘌呤的研究表明，解旋酶和聚合酶都参与了DNA解链，但每一种酶都部分地解链连接碱基对。当结合时，如果解旋酶位于引物末端前面一个核苷酸，则接合碱基对协同解链。联合酶解链平衡的协同变化解释了协同性，其中解旋酶通过促进dNTP结合（降低dNTP Km）刺激聚合酶，聚合酶通过增加解旋速率常数（kcat）刺激解旋酶，因此联合酶解旋DNA的动力学参数类似于单链DNA上的酶易位。http://dx.doi.org/10.7554/eLife.06562.001 DNA复制是一个DNA分子被复制形成两个相同分子的过程。首先，一种叫做DNA解旋酶的酶将DNA双螺旋的两条链分开。这就形成了一种称为复制叉的结构，它有两条暴露的单链。其他称为DNA聚合酶的酶然后使用每条链作为模板来构建新的匹配DNA链。DNA聚合酶通过将称为核苷酸的小分子连接在一起来构建新的DNA链。其中一条新的DNA链--称为“前导链”--是连续构建的，而另一条--“滞后链”--是由一系列短片段组成，然后连接在一起。构建前导链需要解旋酶和DNA聚合酶密切合作。然而，尚不清楚这两种酶如何协调它们的活性。现在，Nandakumar等人已经研究了感染细菌的病毒的解旋酶和DNA聚合酶，并确定了复制叉上酶的确切位置。实验表明，聚合酶和解旋酶都有助于DNA链的分离，并且当解旋酶仅在聚合酶之前的单个核苷酸时，该过程最有效。进一步的实验表明，解旋酶通过帮助聚合酶与核苷酸结合来刺激聚合酶，而聚合酶通过帮助它以更快的速度分离DNA链来刺激解旋酶。下一个挑战是研究允许解旋酶和聚合酶增加彼此活性的分子设置。DOI：http://dx.doi.org/10.7554/eLife.06562.002网站
Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase (DNAP) enzymes, but the structural and mechanistic basis of coupling is poorly understood. This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities. Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting, but each enzyme melts the junction base pair partially. When combined, the junction base pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end. The synergistic shift in equilibrium of junction base pair melting by combined enzymes explains the cooperativity, wherein helicase stimulates the polymerase by promoting dNTP binding (decreasing dNTP Km), polymerase stimulates the helicase by increasing the unwinding rate-constant (kcat), consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA. DOI: http://dx.doi.org/10.7554/eLife.06562.001 DNA replication is the process whereby a molecule of DNA is copied to form two identical molecules. First, an enzyme called a DNA helicase separates the two strands of the DNA double helix. This forms a structure called a replication fork that has two exposed single strands. Other enzymes called DNA polymerases then use each strand as a template to build a new matching DNA strand. DNA polymerases build the new DNA strands by joining together smaller molecules called nucleotides. One of the new DNA strands—called the ‘leading strand’—is built continuously, while the other—the ‘lagging strand’—is made as a series of short fragments that are later joined together. Building the leading strand requires the helicase and DNA polymerase to work closely together. However, it was not clear how these two enzymes coordinate their activity. Now, Nandakumar et al. have studied the helicase and DNA polymerase from a virus that infects bacteria and have pinpointed the exact positions of the enzymes at a replication fork. The experiments revealed that both the polymerase and helicase contribute to the separating of the DNA strands, and that this process is most efficient when the helicase is only a single nucleotide ahead of the polymerase. Further experiments showed that the helicase stimulates the polymerase by helping it to bind to nucleotides, and that the polymerase stimulates the helicase by helping it to separate the DNA strands at a faster rate. The next challenge is to investigate the molecular setup that allows the helicase and polymerase to increase each other's activities. DOI: http://dx.doi.org/10.7554/eLife.06562.002